Key points

Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation of malignant plasma cells in the bone marrow and the development of osteolytic bone lesions. Over the last decade, MM has emerged as a paradigm within the cancers for the success of drug discovery and translational medicine. Despite recent advances in the treatment of MM using conventional and novel therapeutics in combination with transplantation, the disease still remains incurable and most patients eventually relapse. Thus, novel therapeutic approaches, which have a mechanism of action distinct from cytotoxic chemotherapy, are required to eradicate the tumor cells. Immunotherapy is an encouraging option for the goal of inducing effective and long-lasting therapeutic outcome and has become an important approach in the development of treatment strategies for MM. Immunotherapy can be divided into two distinct approaches, passive or active immunotherapies, which target tumor-associated antigens (TAAs) and have shown promising results in multiple preclinical and clinical studies.

Passive-specific immunotherapy

The monoclonal antibodies (mAbs) bind directly to TAA on the surface of myeloma cells, and induce apoptosis directly or trigger antigen-dependent cellular cytotoxicity or complement-dependent cytotoxicity against the tumor cells. A variety of mAbs are undergoing preclinical and clinical investigation in MM. Elotuzumab is a specific mAb directed toward CS1, a glycoprotein that is specific to plasma cells and highly expressed on MM cells, although the antigen may also be expressed in natural killer and CD8 ⁺ T cells. The results in monotherapy were modest ; however, the combination of elotuzumab with lenalidomide and dexamethasone has given excellent results with greater than 80% partial remission in relapsed patients and prolonged progression-free survival. As the proposed mechanism of action, lenalidomide would prepare the natural killer and lymphoid cells by changing the conformation of their cytoskeleton to favor the immune recognition and elotuzumab would modify the plasma cells to be more prone to be targeted by the immune cells. A phase III trial in relapsed myeloma comparing lenalidomide plus dexamethasone with the combination of lenalidomide, dexamethasone, and elotuzumab has recently been completed.

Anti-CD38 mAb (daratumumab, SAR650954, MOR101) has shown consistent cytotoxic activity against MM cells both in vitro and in vivo. Daratumumab as a single agent demonstrated a marked reduction of myeloma cells and bone marrow plasma cells in subjects with relapsed or refractory MM. Remarkably, 42% of the subjects treated achieved partial responses at therapeutic levels in the dose-escalation study with daratumumab monotherapy. This encouraging result has prompted the recent development of other anti-CD38 mAbs, such as SAR650984 and MOR101.

CD40, CD56, and CD138 are other antigens of the plasma cells that have been targeted by mAbs. Lorvotuzumab and nBT062 directed against CD56 and CD138, respectively, have in common that they are each conjugated with a cytotoxic agent (DM1 and DM4, respectively) that is released inside the cells once bound to it. The results of the phase 1 trials in monotherapy showed some minimal responses and partial responses in subjects who were heavily pretreated. mAbs against CD40, dacetuzumab, and lucatumumab have shown some modest responses as monotherapy. Some of these antibodies are currently being combined with other agents, including with lenalidomide and dexamethasone, for a potential immune synergy.

Boost of immune responses has been demonstrated with a mAb specific to programmed cell death 1 (PD-1) on T cells. Blockade of the interaction between PD-1 and PD-1L and effectiveness of anti-PD-1 has also been shown in promoting T-cell activation in MM. Other targets with potential clinical relevance are undergoing investigations such as anti–vascular endothelial growth factor (VEGF) Ab, anti-IL6, or anti-inhibitory killer immunoglobulin–like receptors. A wide variety of monoclonal antibodies ( Table 1 ) are currently under evaluation in clinical trials to treat patients with multiple myeloma.

Passive-specific immunotherapy

The monoclonal antibodies (mAbs) bind directly to TAA on the surface of myeloma cells, and induce apoptosis directly or trigger antigen-dependent cellular cytotoxicity or complement-dependent cytotoxicity against the tumor cells. A variety of mAbs are undergoing preclinical and clinical investigation in MM. Elotuzumab is a specific mAb directed toward CS1, a glycoprotein that is specific to plasma cells and highly expressed on MM cells, although the antigen may also be expressed in natural killer and CD8 ⁺ T cells. The results in monotherapy were modest ; however, the combination of elotuzumab with lenalidomide and dexamethasone has given excellent results with greater than 80% partial remission in relapsed patients and prolonged progression-free survival. As the proposed mechanism of action, lenalidomide would prepare the natural killer and lymphoid cells by changing the conformation of their cytoskeleton to favor the immune recognition and elotuzumab would modify the plasma cells to be more prone to be targeted by the immune cells. A phase III trial in relapsed myeloma comparing lenalidomide plus dexamethasone with the combination of lenalidomide, dexamethasone, and elotuzumab has recently been completed.

Anti-CD38 mAb (daratumumab, SAR650954, MOR101) has shown consistent cytotoxic activity against MM cells both in vitro and in vivo. Daratumumab as a single agent demonstrated a marked reduction of myeloma cells and bone marrow plasma cells in subjects with relapsed or refractory MM. Remarkably, 42% of the subjects treated achieved partial responses at therapeutic levels in the dose-escalation study with daratumumab monotherapy. This encouraging result has prompted the recent development of other anti-CD38 mAbs, such as SAR650984 and MOR101.

CD40, CD56, and CD138 are other antigens of the plasma cells that have been targeted by mAbs. Lorvotuzumab and nBT062 directed against CD56 and CD138, respectively, have in common that they are each conjugated with a cytotoxic agent (DM1 and DM4, respectively) that is released inside the cells once bound to it. The results of the phase 1 trials in monotherapy showed some minimal responses and partial responses in subjects who were heavily pretreated. mAbs against CD40, dacetuzumab, and lucatumumab have shown some modest responses as monotherapy. Some of these antibodies are currently being combined with other agents, including with lenalidomide and dexamethasone, for a potential immune synergy.

Boost of immune responses has been demonstrated with a mAb specific to programmed cell death 1 (PD-1) on T cells. Blockade of the interaction between PD-1 and PD-1L and effectiveness of anti-PD-1 has also been shown in promoting T-cell activation in MM. Other targets with potential clinical relevance are undergoing investigations such as anti–vascular endothelial growth factor (VEGF) Ab, anti-IL6, or anti-inhibitory killer immunoglobulin–like receptors. A wide variety of monoclonal antibodies ( Table 1 ) are currently under evaluation in clinical trials to treat patients with multiple myeloma.

Active-specific immunotherapy

Active-specific immunotherapy has the distinct advantage of inducing highly effective T lymphocytes with antitumor activities and memory functions. Results from recent research have indicated that myeloma cells are susceptible to T cell–mediated cytotoxicity. Long-lasting disease remission has been achieved in patients with MM after infusion of donor lymphocytes in the postallograft relapse setting in which patients are chemotherapy refractory. With the encouraging results of allogeneic transplantation as well as graft-versus-myeloma responses following donor lymphocytes infusion, different types of active-specific immunotherapy approaches are being evaluated to treat patients with MM. Most active-specific immunotherapy protocols in development for MM have used tumor-specific idiotypic protein or whole tumor cells and used dendritic cells (DCs) to generate patient-specific vaccines.

Idiotype (Id) proteins are tumor-specific antigens that can be used for an active immunization against idiotypic determinants on malignant B cells and have been shown to induce resistance to tumor in murine models. Additional studies have shown that T cells in patients with myeloma responded to peptides corresponding to complementarity-determining regions of heavy and light chains of the autologous M-component. In general, most clinical trials conducted using Id-pulsed DCs showed immune responses against the tumor target cells. Interestingly, the Id-induced T-cell stimulation was more confined to the CD4 ⁺ T cell subset than the CD8 ⁺ subset in most of the subjects examined, especially with T _H 2-specific response in subjects with advanced MM (stage II–III). However, the clinical responses were unsatisfactory, mainly due to the poor immunogenicity of the Id protein. To overcome the limitation of weak immunogenicity of Id antigen and to elicit a robust T-cell response, several Id vaccination strategies have been adopted: immunization of the purified M component together with adjuvant cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), keyhole-limpet-hemocyanin-coupled paraprotein immunization, and Id-loaded DC administration. In addition, different DC sources such as Langerhans cells could be explored because they have been shown to be comparable to monocytes-derived DC in generating effector T cells specific to tumor cells. More recent results demonstrated improved clinical response by DC-based Id vaccination. A commercial product is currently being tested in phase III trial. Mylovenge (APC8020) is conducted by pulsing autologous DC with the subject’s Id and showed that the long-term survival in the subjects with MM who received the vaccine and underwent autologous hematopoietic stem cell transplant.

An alternative DC-based vaccination approach involves the fusion of autologous DC with patient-derived tumor cells to enhance the immunogenicity of tumor antigens. DC fusion cells can stimulate both helper and cytotoxic T-cell responses through the presentation of internalized and newly synthesized antigens. Lenalidomide has been shown to further improve the immunogenicity of DC-MM fusion vaccines by polarizing T-cell responses into Th1 and reducing regulatory T cells and the expression of immune suppressive molecules on T cells. Recently, phase II studies were undertaken in which subjects with MM were vaccinated with an autologous DC-tumor cell fusion in combination with GM-CSF administration on the day of DC vaccination following autologous stem cell transplantation (ASCT) to target minimal residual disease. In the study, the second cohort of 12 subjects received a pretransplant vaccine followed by posttransplant vaccinations. The posttransplant period was associated with reduction in general measures of cellular immunity; however, an increase in myeloma-specific CD4 ⁺ and CD8 ⁺ T cells was observed after ASCT and the specific effector cells were significantly expanded following posttransplant vaccination. A high proportion (78%) of MM patients achieved either a complete response or very good partial response following vaccination, thereby demonstrating the potential for a positive clinical outcome using vaccine immunotherapy to treat multiple myeloma.

In addition, whole tumor cells or tumor lysates have been used to improve the efficacy of the DC-based vaccine in subjects with MM. There have been increasing reports of alternative approaches such as DC pulsed with tumor lysates, apoptotic tumor cells, or DC transfected with myeloma-derived RNA. Apoptotic bodies were shown to be more effective than cell lysate at inducing cytotoxic T lymphocytes (CTLs) against autologous myeloma cells. In addition, tumor-derived heat shock proteins (HSPs), such as HSP70 and gp96, demonstrated their immunogenic characteristics and the myeloma-derived gp96-loaded DC were used to generate tumor-specific CTLs that were able to lyse tumor cells in patients with MM in an major histocompatibility complex (MHC) class I–restricted manner.

However, these approaches with patient-specific protocols are labor-intensive and cost-ineffective, making their general applicability challenging. To overcome this limitation, development of an off-the-shelf–based immunotherapy is necessary for treating patients more efficiently. Among several options, peptide-based vaccines offer distinct advantages over individualized vaccines with regards to safety, broader applicability, low toxicity, easy of production, and monitoring for tumor-specific immune response in patients. In addition, this approach can successfully induce antitumor immune responses with the potential of epitope spreading, whereby lysed target cancer cells release new antigenic epitopes, which are then taken up, processed, and presented by antigen-presenting cells to a new repertoire of CTL, and thereby further tumor lysis. Although there is MHC restriction in this peptide-specific vaccine approach, application of cocktails of immunogenic peptides to different HLA molecules would broaden the induction of CTL specific to tumor cells of multiple MHC classifications. Based on the recent progress on the discovery of TAA, epitopes have been identified from multiple potential antigens and evaluated for the development of vaccines by eliciting the antigen-specific CD8 ⁺ T cell responses against MM cells. Strategies for further improvement in the efficacy of therapy, including combined use of chemotherapy drugs and molecular target-based drugs, are being proposed. Peptide vaccination in an “adjuvant setting” should be considered a promising treatment to protect against progression or relapse of malignancies with minimal residual disease.

Following are several types of TAA applied and progress made for the development of peptide-based vaccines in MM.

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  X-box binding protein 1 (XBP1), a critical transcription activator in the unfolded protein response, regulates a subset of endoplasmic reticulum–resident chaperone genes essential for protein folding and maturation. Genome-wide profiling, along with association studies and immunohistochemistry, demonstrated that XBP1 expression was induced in a variety of cancers, including hematological malignancies such as MM and solid tumors. It has been reported that XBP1 is activated in primary mammary tumors, that its expression correlates with enhanced tumor growth, moreover, transformed cells with XBP1 deficiency were sensitized to hypoxia and underwent apoptosis, implicating XBP1 as a survival factor. Thus, disrupting or targeting the XBP1 pathway is a rational approach for selective cancer cell killing, providing the basis for therapeutic strategies against multiple solid tumors. In MM, it is highly expressed in primary cells and cell lines, selectively induced by exposure to IL-6, and has been implicated in the proliferation of malignant plasma cells. Based on these observations, Bae and colleagues proposed the XBP1 as a unique therapeutic target antigen and identified two heteroclitic peptides, YISPWILAV and YLFPQLISV, with improved HLA-A2-binding and stability from their respective native peptides, XBP1 _184−192 (NISPWILAV) and XBP1 SP _367−375 (ELFPQLISV). The XBP1 peptides-specific CTL showed distinct phenotypes and functional activities and demonstrated MM-specific and HLA-A2-restricted proliferation, interferon (IFN)-γ secretion, and/or cytotoxic activity in response to MM cell lines and primary MM cells. These data demonstrate the distinct immunogenic characteristics of unique heteroclitic XBP1 peptides, which induce MM-specific CTL.

  
  
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  The CD138, also known as syndecan-1, is a transmembrane heparan sulfate–bearing proteoglycan expressed by most MM cells. CD138 is critical for the growth of tumor cells by mediating cell-cell adhesion, binding MM cells to molecules such as collagen and fibronectin in the extracellular matrix, as well as binding to growth factors and cytokines. In patients with MM, shed syndecan-1 accumulates in the bone marrow, and soluble syndecan-1 facilitates MM tumor progression, angiogenesis, and metastasis in vivo. It has a cytoplasmic domain that is linked to cytoskeletal elements to potentiate anchorage of the cells and stabilize cell morphology, whereas their extracellular domain has up to three heparan sulfate chains that bind to numerous soluble and insoluble molecules, thus CD138 has critical roles for the growth of tumor cells. Therefore, targeting CD138 on malignant plasma cells to prevent or reduce high levels of syndecan-1 in the serum, an indicator of poor prognosis in MM, may have a direct clinical benefit. A novel immunogenic HLA-A2-specific peptide, CD138 _260-268 (GLVGLIFAV), identified by Bae and colleagues, induces antigen-specific CTL, and the CD138 peptide-specific CTL displayed a unique immunologic phenotype, as well as HLA-A2-restricted responses and functional activities against both primary MM cells and MM cell lines expressing CD138 antigen.

  
  
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  CS1 is a cell surface glycoprotein of the CD2 family, which is highly and uniformly expressed by malignant plasma cells and has restricted expression in normal tissues. CS1 localizes to the uropods of polarized MM cells, where it mediates adhesion of MM cells to bone marrow stroma and other human MM cells. CS1 expression was observed on MM cells from all subjects, including MM with high-risk and low-risk molecular profiles, and those with and without cytogenetic abnormalities, suggesting that this antigen is not restricted to any particular MM subgroup. Equally important for the development of immunotherapy, CS1 expression is maintained on subjects’ MM cells even after relapse of disease. Based on these findings, Bae and colleagues identified a novel immunogenic HLA-A2-specific epitope, CS1 _239-247 peptide (SLFVLGLFL), which is derived from the CS1 antigen and has the ability to evoke MM-specific CTL. With the findings of universal expression of these functional antigens on MM cells, the development of an immunotherapeutic strategy targeting XBP1, CD138, and CS1 antigens was proposed as a novel treatment option for MM, and the multipeptide was evaluated for its immunogenicity as a cocktail to induce the peptides-specific CTL from smoldering MM (SMM) subjects’ T cells. The multipeptide-specific CTL generated from SMM subjects’ T cells demonstrated dramatic phenotypic changes and effective anti-MM responses, including the upregulation of critical markers including CD69 and CD137 (4-1BB), CTL proliferation, IFN-γ production, and degranulation (CD107a) in an HLA-A2-restricted and peptide-specific manner. The results also suggest that this multipeptide cocktail has the potential to induce effective and durable memory membraneproteoglycan-CTL in SMM subjects. Therefore, these findings provide the rationale for clinical evaluation of a therapeutic vaccine to prevent or delay progression of SMM to active disease. Clinical applicability of the peptides derived from these antigens is undergoing evaluation.

  
  
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  Dickkopf-1 (DKK1) is a secreted protein that specifically inhibits the Wnt/[beta]-catenin signaling by interacting with the coreceptor Lrp-6. In addition to its direct inhibitory effect of DKK1 on osteoblasts, DKK1 disrupts the Wnt3a-regulated osteoprotegerin and receptor activator of nuclear factor–kappaB ligand (RANKL) expression in osteoblasts and thus it indirectly enhances osteoclast function in MM. Recent studies demonstrated that DKK1 in subjects with myeloma was associated with the presence of lytic bone lesions and DKK1 plays an important role in myeloma bone disease. Qian and colleagues identified HLA-A2-specific peptides derived from DKK1 that was capable of inducing DKK1-specific T-cell lines and clones from HLA-A2 ⁺ normal donors and MM patients. These CTL showed peptide-specific and MM-specific responses in vitro and showed the therapeutic efficacy in vivo against established tumor cells in an HLA-A2 transgenic murine model. They detected low frequencies of DKK1 peptide-specific CD8 ⁺ T cells in subjects with myeloma by using tetramers, peptide-specific T-cell lines, and clones generated from HLA-A2 ⁺ blood donors or patients with myeloma. These T cells efficiently lysed peptide-pulsed but not unpulsed T2 or autologous DC, DKK1 ⁺ /HLA-A2 ⁺ myeloma cell lines U266 and IM-9 as well as HLA-A2 ⁺ primary myeloma cells from patients. Thus, these data show that DKK1 is a novel target for the management of myeloma patients with lytic bone disease.

  
  
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  Receptor for hyaluronic acid mediated motility (RHAMM) is another immunogenic antigen that plays a critical role in tumorigenesis. It is highly expressed in hematological malignancies including MM and induces humoral and cellular immune responses. RHAMM-R3 peptide has been identified and investigated as a vaccine in subjects with MM (Schmitt and colleagues and Greiner and colleagues ). In their phase I trial, the RHAMM-R3 peptide (ILSLELMKL) was administered four times (300 μg or 1 mg/vaccination) subcutaneously at a biweekly interval to HLA-A2 ⁺ MM subjects who were in partial remission or near complete remission after high-dose chemotherapy with melphalan and autologous stem cell transplantation. Encouraging immune monitoring results were detected: (1) an increase (>50%) in IFN-γ ⁺ and granzyme ⁺ spots in ELISPOT analyses, (2) an increase (>50%) in HLA-A2/R3-tetramer ⁺ /CD8 ⁺ T lymphocytes and with an increase (>25%) in RHAMM-R3-tetramer ⁺ /CD8 ⁺ T lymphocytes, and (3) CD8 ⁺ T cell responsiveness. The MM patients who had a positive immune response showed an increase of CD8 ⁺ tetramer ⁺ /CD45RA ⁺ /CCR7 ⁻ /CD27 ⁻ /CD28 ⁻ effector T cells and RHAMM-R3-specific CD8 ⁺ T cells. In this study, 50% (2 out of 4) subjects with MM showed a reduction of free light chain serum levels. High-dose RHAMM-R3 peptide vaccination induced positive clinical effects.

  
  
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  Cancer testis antigens (CTAs) have been extensively investigated in MM for their expression and application as target antigens. DNA microarray analysis of gene expression of greater than 95% pure myeloma cells from more than 300 subjects showed that the genes of MAGE-3 and NY-ESO-1 antigens were expressed in the tumor cells, particularly from subjects with relapsed disease or abnormal cytogenetics (in 7%–20% of MGUS and newly diagnosed MM and in 40%–50% of relapsed subjects or in subjects with cytogenetic abnormalities). In addition, the protein expression of these antigens were also demonstrated in the tumor cells of subjects with positive gene expression. The mechanisms that underlie this expression are unclear but are at least partially related to demethylation of gene promoter sequences. The HLA-A1-restricted or HLA-A2-restricted MAGE-3- or NY-ESO-1-specific peptide have been identified and the tumor-specific CTL generated by the peptide were demonstrated against myeloma cells. In addition, other antigens, such as MUC-1, sperm protein 17 (Sp17), and HM1.24 may also be expressed on myeloma cells, and MHC-restricted antigen-specific CTL have been generated from subjects with myeloma that were able to lyse myeloma cells. Recently, a phase I–II clinical trial has been initiated to examine the safety and efficacy of Sp17-pulsed DC vaccination in subjects with myeloma. Anderson and colleagues identified peptides derived from MAGE-C1 (CT-7), which is the most commonly expressed CTA found in MM. The CT-7-specific CTL recognizing two peptides targeted both MM cells as well as CT-7 gene-transduced tumor cells. They demonstrated that these epitopes are promising targets for developing an immunotherapy against myeloma or other CT-7 ⁺ malignancies. Clinical applicability of the peptides derived from the cancer testis antigens are undergoing evaluation.

The current clinical trials using active-specific immunotherapy are shown in Table 2 to treat the patients with multiple myeloma.

Table 2

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